1. Field
Embodiments of the present invention relate to fluorescence analysis systems and in particular to light emitting diode (LED)-based fluorescence analysis systems.
2. Discussion of Related Art
Fluorescence is the emission of light by molecules which have absorbed light. The fluorescing characteristics of such molecules (called fluorophors) are useful in detecting and tagging various microbiological events. The emission signal is shifted to higher wavelengths (Stokes-Shift) in relation to the excitation signal because the energy of the fluorescing light emitted is less than the light absorbed by the fluorophor.
Fluorometers exploit the fluorescing property of fluorophor molecules in the analysis of biological samples. The simulation and optimization of a LED-based fluorometer should ideally maximize the efficiency with which light is converted to signal (emission) and minimize bleed-through excitation light to the output signal path. In other words, the overlap between excitation light source and emission spectra should be minimized so that once the excitation light is optically filtered from the output signal, maximum emission signal remains for measurement.
Conventional fluorescence analysis systems use a laser or a high power white light source (e.g. Xenon lamp) to excite fluorophors in the sample under analysis. LEDs (light emitting diodes) are of interest to replace conventional light sources to increase the portability (reduce power, size, and weight) of the analysis system and to improve the flexibility of excitation spectra available to the user with reduced optics overhead. Several approaches have been used in LED-based fluorescence analysis systems. LEDs are an attractive alternative to conventional white light sources used in fluorescence analysis because of reduced power of operation, fewer imaging artifacts, and increased flexibility in spectral control without the need for high overhead optics.
General purpose, commercially available portable systems, such as the Turner Biosystems [1][2] instruments have used single LEDs to excite fluorescing samples; these systems rely on the user to select the LED to match the fluorophor or vice versa. Many results in the literature rely on single or small arrays of LEDs, where excitation bands are chosen close to the excitation spectra of the fluorophor and the resulting emission spectrum is optically filtered to minimize interference from the excitation signal. Still other approaches excite a sample using different LEDs at different times and subsequent signal processing to improve the extraction of the emission signal from the combined output signal. Finally, a variety of waveguides have been constructed to minimize the transfer of excitation light along the output emission path at the expense of reduced sample volume.
The use of LEDs, however, is often limited by three primary factors: (a) the broadband output of an LED often interferes with the measurement of emission signal; (b) the power (intensity) of light generated by an LED (mWatts) is often small compared to white light source (Watts) counterparts; and (c) the excitation peaks of the LED are often not well matched to the absorption efficiency of the fluorophor under analysis. The use of LEDs, for this reason, has been largely limited to high concentration applications where emitted fluorescence is sufficiently high (and noise sufficiently low) that LED limitations do not restrict effective measurements of the sample under analysis. The spectral flexibility, modularity, low-cost, and low power consumption of LEDs, however, continue to make them attractive options for fluorescence analysis, however.
In many approaches using LEDs, the choice of LED (or LEDs) is usually not optimized prior to the collection of data by the fluorescence analysis system. Instead, the optics and signal processing are assigned the task of separating excitation components from the emission signal in the output path. In addition, many LED-based fluorescence analysis systems used in commercial and research efforts are general-purpose. This means that they are suited to a relatively wide selection of fluorophors and the biological applications to which they are applied.